U.S. patent number 6,287,451 [Application Number 09/324,443] was granted by the patent office on 2001-09-11 for disposable sensor and method of making.
Invention is credited to Xiaohua Cai, Fung Seto, Handani Winarta, Chung Chang Young.
United States Patent |
6,287,451 |
Winarta , et al. |
September 11, 2001 |
Disposable sensor and method of making
Abstract
A disposable electrode strip for testing a fluid sample
including a laminated strip with a first and second end, a
reference electrode embedded in the laminated strip proximate to
the first end, at least two working electrodes embedded in the
laminated strip proximate to the first end and the reference
electrode, an open path for receiving a fluid sample beginning from
the first end and being sufficiently long to expose the reference
electrode and the working electrodes to the fluid sample, and
conductive contacts located at the second end of the laminated
strip. The laminated strip has a base layer with a conductive
coating, a reagent holding layer, a channel forming layer and a
cover. One of the working electrodes contains a reagent
substantially similar to the reagent of the reference electrode and
a second working electrode contains a reagent having an enzyme.
Inventors: |
Winarta; Handani (Nashua,
NH), Cai; Xiaohua (Needham, MA), Seto; Fung (Newton,
MA), Young; Chung Chang (Weston, MA) |
Family
ID: |
23263613 |
Appl.
No.: |
09/324,443 |
Filed: |
June 2, 1999 |
Current U.S.
Class: |
205/777.5;
205/792; 216/52; 204/403.11; 204/403.03; 204/403.14 |
Current CPC
Class: |
G01N
27/3272 (20130101); C12Q 1/002 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101); G01N 027/26 () |
Field of
Search: |
;204/403,412
;205/777.5,775,792 ;216/52 ;438/460,462,463 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0096095 |
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Dec 1983 |
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EP |
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0121385 |
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Oct 1984 |
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EP |
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0136362 |
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Apr 1985 |
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EP |
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0170375 |
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Feb 1986 |
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EP |
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0255291 |
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Feb 1988 |
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EP |
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41111879 A |
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Apr 1999 |
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JP |
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8803270 |
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May 1988 |
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WO |
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Other References
JPAB abstract of Miyamoto et al.. (JP 411118794A), Apr. 1999.*
.
WO 98/35225 PCT Application, Aug. 13, 1998, Adam Heller et al.
.
WO 98/55856 PCT Application, Dec. 10, 1998, Stephen Williams et
al..
|
Primary Examiner: Tung; T.
Assistant Examiner: Noguerola; Alex
Attorney, Agent or Firm: DeLeault, Esq.; Robert R. Mesmer
& DeLeault, PLLC
Claims
What is claimed is:
1. A disposable electrode strip for testing a fluid sample
comprising:
a laminated strip having a first strip end, a second strip end and
a vent opening spaced from said first strip end, said laminated
strip comprising a base layer with at least three electrodes
delineated thereon, a reagent holding layer carried on said base
layer, said reagent holding layer having at least three cutouts, a
channel forming layer carried on said reagent holding layer, and a
cover;
an enclosed channel between said first strip end and said vent
opening, said enclosed channel containing said at least three
cutouts;
a first reagent disposed in a first cutout of said at least three
cutouts forming a reference electrode;
a second reagent disposed in a second cutout of said at least three
cutouts forming a first working electrode, said second reagent
being substantially similar to said first reagent;
a third reagent disposed in a third cutout of said at least three
cutouts forming a second working electrode, said third reagent
containing an enzyme; and
conductive contacts at said second strip end and insulated from
said enclosed channel.
2. The electrode strip of claim 1 wherein said enzyme is glucose
oxidase.
3. The electrode strip of claim 1 wherein said first reagent, said
second reagent and said third reagent contain a redox mediator.
4. The electrode strip of claim 3 wherein said first reagent, said
second reagent and said third reagent further contain at least one
of a stabilizer, a binder, a surfactant, and a buffer.
5. The electrode strip of claim 4 wherein said stabilizer is a
polyalkylene glycol, said binder is a cellulose material, and said
surfactant is a polyoxyethylene ether.
6. The electrode strip of claim 5 wherein said stabilizer is
polyethylene glycol, said binder is methyl cellulose, said
surfactant is t-octylphenoxypolyethoxyethanol, and said buffer is a
citrate buffer.
7. The electrode strip of claim 6 wherein said first reagent and
said second reagent are made of a mixture having starting
components comprising about 1 wt % of said potassium ferricyanide,
about 2.5 wt % of said polyethylene glycol, about 1 wt % of said
methyl cellulose, about 0.03 wt % of said
t-octylphenoxypolyethoxyethanol, and said citrate buffer is about
0.05M.
8. The electrode strip of claim 6 wherein said third reagent is
made of a mixture having starting components comprising about 6.5
wt % of said potassium ferricyanide, about 2.5 wt % of said
polyethylene glycol, about 1 wt % of said methyl cellulose, about
0.03 wt % of said t-octylphenoxypolyethoxyethanol, and said pH
buffer is about a 0.05M citrate buffer, and about 1 wt % of said
enzyme.
9. The electrode strip of claim 8 wherein said enzyme is glucose
oxidase.
10. The electrode strip of claim 4 wherein said first reagent, said
second reagent and said third reagent are made from a mixture
having starting components comprising about 1 wt % to about 6.5 wt
% of said redox mediator, about 2.5 wt % of said stabilizer, about
1 wt % of said binder, and about 0.03 wt % of said surfactant in
said buffer.
11. The electrode strip of claim 10 wherein said citrate buffer is
about 0.05M.
12. The electrode strip of claim 3 wherein said redox mediator is
at least one metal complex.
13. The electrode strip of claim 3 wherein said at least one redox
mediator is selected from the group consisting of potassium
ferricyanide and other inorganic and organic redox mediators.
14. The electrode strip of claim 1 wherein said base layer has a
conductive coating disposed thereon for forming said at least three
electrodes.
15. The electrode strip of claim 14 wherein said conductive coating
is gold.
16. The electrode strip of claim 14 wherein said conductive coating
comprising gold and tin oxide.
17. The electrode strip of claim 14 wherein said base layer, said
reagent holding layer, said channel forming layer, and said cover
are made of a plastic dielectric material.
18. The electrode strip of claim 17 wherein said plastic material
is selected from the group consisting of polyvinyl chloride,
polycarbonate, polysulfone, nylon, polyurethane, cellulose nitrate,
cellulose propionate, cellulose acetate, cellulose acetate
butyrate, polyester, acrylic, and polystyrene.
19. The electrode strip of claim 1 wherein said enclosed channel is
hydrophilic.
20. The electrode strip of claim 1 wherein said enclosed channel
has a volume of about 1.44 microliters.
21. The electrode strip of claim 1 wherein said cover has a
hydrophilic coating on at least one side.
22. The electrode strip of claim 1 wherein said channel forming
layer has a thickness sufficient to optimize the flow of said fluid
sample along said open path.
23. The electrode strip of claim 22 wherein said thickness is about
0.007 inches (0.1778 mm).
24. The electrode strip of claim 1 wherein the surface area of said
first working electrode is substantially same as the surface area
of said second working electrode.
25. A method of using an electrode strip for determining the
concentration of an analyte, said electrode strip having a first
working electrode, a second working electrode and a reference
electrode wherein said first working electrode contains an enzyme
capable of catalyzing a reaction involving a substrate for the
enzyme, said first working electrode, said second working electrode
and said reference electrode being disposed in a fluid sample
channel for measuring a fluid sample, said method comprising:
disposing said fluid sample into said channel of said electrode
strip;
applying a potential between said reference electrode and said
first working electrode which contains said enzyme;
measuring a first current generated between said first working
electrode and said reference electrode and correlating said first
current to a concentration of said analyte in said fluid
sample;
measuring a resistance value of said fluid sample between said
second working electrode and said reference electrode;
applying said resistance value to a first equation and determining
the hematocrit level of said fluid sample; and
calculating a corrected concentration of said analyte using a
second equation to correct for the presence of hematocrit in said
sample.
26. The method of claim 25 wherein said method further
comprising:
applying a potential between said reference electrode and said
second working electrode;
measuring a second current generated between said second working
electrode and said reference electrode;
subtracting said second current from said first current and
obtaining a current difference, correlating said current difference
to a concentration of said analyte in said fluid sample.
27. The method of claim 26 wherein said method further includes
triggering said current measuring step when said fluid sample
contacts said first working electrode, said second working
electrode and said reference electrode creating said first current
and said second current.
28. The method of claim 27 wherein said method further includes
reading a current value for each of said first current and said
second current at about a time where said current values for each
of said first current and said second current reach a
steady-state.
29. The method of claim 28 wherein said reading is taken at about
20 seconds after said current measuring step is triggered.
30. A disposable electrode strip for detecting or measuring the
concentration of at least one analyte in a fluid sample, said
electrode strip comprising:
an insulating base strip having a first base end and a second base
end;
a conductive layer disposed on one side of said base strip
delineating at least three electrically-distinct conductive paths
insulated from each other;
a first electrical insulator sized smaller than said insulating
base strip and overlaying a substantial portion of said conductive
layer, said first insulator having at least a first cutout portion
and a second cutout portion spaced from said first base end, said
first cutout portion exposing a limited area of a first of
said at least three conductive paths and said second cutout portion
exposing a limited area of a second and a third of said at least
three conductive paths;
at least two electrode materials wherein a first material of said
at least two electrode materials is a reagent for measuring the
concentration of said at least one analyte and wherein a second
material of said at least two electrode materials is a material
suitable for use as a reference material and for measuring the
resistance of said fluid sample, said first material being disposed
in said first cutout potion and said second material being disposed
in said second cutout portion, said second material being scored to
isolate said second material disposed on said second of said at
least three conductive paths from said second material disposed on
said third of said at least three conductive paths; and
a second electrical insulator sized to fit over and coextensive
with said first electrical insulator, said second insulator having
an opening configured to expose an area of said first insulator a
limited distance from said first base end of said insulating base
strip, said area including said at least two cutout portions of
said first insulator; and
a third electrical insulator sized to fit over and coextensive with
said second insulator creating a sample fluid channel, said third
insulator having a third insulator vent aperture spaced from said
first base end and configured to expose at least a small portion of
said opening of said second insulator.
31. The strip of claim 30 wherein said sample fluid channel has a
volume of 1.44 microliters.
32. The strip of claim 30 wherein said sample fluid channel is
hydrophilic.
33. The device of claim 30 wherein said first material and said
second material are mixtures having starting components comprising
a redox mediator, a stabilizer, a binder, a surfactant, and a
buffer.
34. The strip of claim 33 wherein said redox mediator is at least
one metal complex selected from the group consisting of ferrocene,
ferrocene derivatives and potassium ferricyanide, said stabilizer
is a polyalkylene glycol, said binder is a cellulose material, said
surfactant is a polyoxyethylene ether, and said buffer has a pH of
about 5 to about 6.
35. The strip of claim 34 wherein said mediator is potassium
ferricyanide, said stabilizer is polyethylene glycol, said binder
is methyl cellulose, said surfactant is
t-octylphenoxypolyethoxyethanol, and said buffer is a citrate
buffer.
36. The strip of claim 35 wherein said first material is made of a
mixture having starting components comprising about 1 wt % of said
potassium ferricyanide, about 2.5 wt % of said polyethylene glycol,
about 1 wt % of said methyl cellulose, and about 0.03 wt % of said
t-octylphenoxypolyethoxyethanol in said citrate buffer.
37. The strip of claim 35 wherein said second material is made of a
mixture having starting components comprising about 6.5 wt % of
said potassium ferricyanide, about 2.5 wt % of said polyethylene
glycol, about 1 wt % of said methyl cellulose, about 0.03 wt % of
said t-octylphenoxypolyethoxyethanol, and about 1 wt % of an enzyme
in said citrate buffer.
38. The strip of claim 37 wherein said enzyme is glucose
oxidase.
39. The strip of claim 30 wherein said insulating base strip, said
first electrical insulator, said second electrical insulator, and
said third electrical insulator are made from a plastic material
selected from the group consisting of polyvinyl chloride,
polycarbonate, polysulfone, nylon, polyurethane, cellulose nitrate,
cellulose propionate, cellulose acetate, cellulose acetate
butyrate, polyester, acrylic, and polystyrene.
40. A method of making multiple, disposable sensors wherein each
sensor has a first working electrode, a second working electrode
and a reference electrode, wherein said first working electrode
contains an enzyme capable of catalyzing a reaction involving a
substrate for the enzyme, said first working electrode, said second
working electrode and said reference electrode being disposed in a
fluid sample channel for measuring a fluid sample, said method
comprising:
obtaining a base strip of an insulating material having a layer of
conductive material disposed thereon, said base strip having a
first edge and a second edge;
scribing in said conductive material a plurality of lines in a
repetitive pattern wherein said plurality of lines contain a
repetitive pattern forming three conductive paths in each of said
repetitive pattern;
disposing a first middle layer of insulating material over said
base strip, said first middle layer having a repetitive pattern of
three cutouts wherein each cutout of each of said repetitive
pattern exposes an electrode portion of each of said three
conductive paths of each repetitive pattern wherein said repetitive
pattern of said three cutouts are spaced from said first edge of
said base strip, and wherein said first middle layer is sized to
expose a contact portion of each of said three conductive paths of
each repetitive pattern for a distance from said second edge of
said base strip;
disposing a first reagent material on two of said three cutouts of
each repetitive pattern and a second reagent material on the other
of said three cutouts of each repetitive pattern;
drying said first reagent material and said second reagent
material;
overlaying a second middle layer of insulating material over and
coextensive with said first middle layer; said second middle layer
having a plurality of elongated cutout portions in a repetitive
pattern wherein each of said elongated cutout portions exposes a
corresponding repetitive pattern of said three cutouts said first
middle layer;
disposing a top layer of insulating material over and coextensive
with said second middle layer, said top layer having a plurality of
vent openings in a repetitive pattern wherein each of said vent
openings exposes a portion of a corresponding repetitive pattern of
said elongated cutout portion furthest from said first edge of said
base strip; and
separating each of said repetitive pattern forming one of each of
said disposable sensors.
41. The method of claim 40 further comprising drying said first
reagent material and said second reagent material at a temperature
and for a length of time sufficient to allow said first reagent
material and said second reagent material to solidify and adhere to
each of said electrode portion of each of said repetitive pattern
of said three conductive paths.
42. The method of claim 40 further comprising cutting along said
first edge of each of said sensors and transverse to said sensors a
predetermined distance creating a sample inlet port.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to electrochemical sensors
that can be used for the quantification of a specific component or
analyte in a liquid sample. Particularly, this invention relates to
a new and improved electrochemical sensor and to a new and improved
method of fabricating electrochemical sensors. More particularly,
this invention relates to a disposable electrochemical sensor that
is inexpensive to manufacture. Even more particularly, this
invention relates to a disposable electrochemical sensor that gives
accurate readings in the presence of interferants and varying red
blood cells (hematocrit). Still even more particularly, this
invention relates to disposable electrochemical sensors which are
used for performing electrochemical assays for the accurate
determination of analytes in physiological fluids.
2. Description of the Prior Art
Biosensors have been known for more than three decades. They are
used to determine concentrations of various analytes in fluids. Of
particular interest is the measurement of blood glucose. It is well
known that the concentration of blood glucose is extremely
important for maintaining homeostasis. Products that measure
fluctuations in a person's blood sugar, or glucose levels have
become everyday necessities for many of the nation's millions of
diabetics. Because this disorder can cause dangerous anomalies in
blood chemistry and is believed to be a contributor to vision loss
and kidney failure, most diabetics need to test themselves
periodically and adjust their glucose level accordingly, usually
with insulin injections. If the concentration of blood glucose is
below the normal range, patients can suffer from unconsciousness
and lowered blood pressure which may even result in death. If the
blood glucose concentration is higher than the normal range, the
excess blood glucose can result in synthesis of fatty acids and
cholesterol, and in diabetics, coma. Thus, the measurement of blood
glucose levels has become a daily necessity for diabetic
individuals who control their level of blood glucose by insulin
therapy.
Patients who are insulin dependent are instructed by doctors to
check their blood-sugar levels as often as four times a day. To
accommodate a normal life style to the need of frequent monitoring
of glucose levels, home blood glucose testing was made available
with the development of reagent strips for whole blood testing.
One type of blood glucose biosensors is an enzyme electrode
combined with a mediator compound which shuttles electrons between
the enzyme and the electrode resulting in a measurable current
signal when glucose is present. The most commonly used mediators
are potassium ferricyanide, ferrocene and its derivatives, as well
as other metal-complexes. Many sensors based on this second type of
electrode have been disclosed. Examples of this type of device are
disclosed in the following patents.
U.S. Pat. No. 5,628,890 (1997, Carter et al.) Disclosed an
electrode strip having an electrode support, a reference or counter
electrode disposed on the support, a working electrode spaced from
the reference or counter electrode on the support, a covering layer
defining an enclosed space over the reference and working
electrodes and having an aperture for receiving a sample into the
enclosed space, and a plurality of mesh layers interposed in the
enclosed space between the covering layer and the support. The
covering layer has a sample application aperture spaced from the
electrodes. The working electrode includes an enzyme capable of
catalyzing a reaction involving a substrate for the enzyme and a
mediator capable of transferring electrons between the
enzyme-catalyzed reaction and the working electrode.
This device proposes to reduce the effect of hematocrit on the
sensor readings. According to the disclosure, this results from the
downstream spacing of the reference electrode relative to the
working electrode in combination with the thin layer of the sample
solution created by the mesh layers.
U.S. Pat. No. 5,708,247 (1998, McAleer et al.) Disclosed a
disposable glucose test strip having a substrate, a reference
electrode, a working electrode, and a means for making an
electrical connection. The working electrode has a conductive base
layer and a coating layer disposed over the conductive base layer.
The coating layer is a filler having both hydrophobic and
hydrophilic surface regions which form a network, an enzyme and a
mediator.
U.S. Pat. No. 5,682,884 (1997, Hill et al.) Disclosed a strip
electrode with screen printing. The strip has an elongated support
which includes a first and second conductor each extending along
the support. An active electrode, positioned to contact the liquid
mixture and the first conductor, has a deposit of an enzyme capable
of catalyzing a reaction and an electron mediator. A reference
electrode is positioned to contact the mixture and the second
conductor.
U.S. Pat. No. 5,759,364 (1998, Charlton et al.) Disclosed an
electrochemical biosensor having an insulating base plate bearing
an electrode on its surface which reacts with an analyte to produce
mobile electrons. The base plate is mated with a lid of deformable
material which has a concave area surrounded by a flat surface so
that when mated to the base plate there is formed a capillary space
into which a fluid test sample can be drawn. The side of the lid
facing the base is coated with a polymeric material which serves to
bond the lid to the base plate and to increase the hydrophilic
nature of the capillary space.
U.S. Pat. No. 5,762,770 (1998, Pritchard et al.) Disclosed an
electrochemical biosensor test strip that has a minimum volume
blood sample requirement of about 9 microliters. The test strip has
a working and counter electrodes that are substantially the same
size and made of the same electrically conducting material placed
on a first insulating substrate. Overlaying the electrodes is a
second insulating substrate which includes a cutout portion that
forms a reagent well. The cutout portion exposes a smaller area of
the counter electrode than the working electrode. A reagent for
analysis of an analyte substantially covers the exposed areas of
the working and counter electrodes in the reagent well. Overlaying
the reagent well and affixed to the second insulating substrate is
a spreading mesh that is impregnated with a surfactant.
U.S. Pat. No. 5,755,953 (1998, Henning et al.) Disclosed an
reduced-interference biosensor. The device generally comprises an
electrode used to electrochemically measure the concentration of an
analyte of interest in a solution. The device includes a peroxidase
enzyme covalently bound to microparticle carbon and retained in a
matrix in intimate contact with the electrode. According to this
disclosure, it is the enzyme/microparticle carbon of the device
which provides a composition which is displays little sensitivity
to known interfering substances.
U.S. Pat. No. 5,120,420 (1992, Nankai et al.) Disclosed a biosensor
with a base board having an electrode system mainly made of carbon,
an insulating layer, a reaction layer containing an enzyme layer
thereon, a spacer and a cover. The spacer creates a channel with an
inlet and an outlet for holding a sample.
However, the prior art devices suffer from various shortcomings.
One of these shortcomings is interference with biosensor readings
caused by other substances in the sample fluid which can oxidize at
the same potential. Prevalent among these are ascorbic acid, uric
acid and acetaminophen. As these and other interfering substances
oxidize, the current resulting from their oxidation is added to and
indistinguishable from the current resulting from the oxidation of
the blood analyte being measured. An error therefore results in the
quantification of the blood analyte.
Another shortcoming is the interference caused by red blood cells
(the hematocrit effect). This interference tends to cause an
artificially high response rate for low hematocrit levels and,
conversely, an artificially low response rate for high hematocrit
levels.
Additional shortcomings of the prior art devices are that they have
a more limited linear range and require a relatively large quantity
of sample volume. Further, they require a relatively longer waiting
time for development of a steady-state response before a reading
can be achieved. Each of these shortcomings may, either
individually or when combined with one or more of the other
shortcomings, contribute to erroneous measurement readings during
analysis. Preliminary tests performed by the inventors of the
present invention have shown that the prior art which claims to
reduce the effect of hematocrit on glucose readings, were limited
to and worked only in lower glucose concentrations.
Because of the importance of obtaining accurate glucose readings,
it would be highly desirable to develop a reliable and
user-friendly electrochemical sensor which does not have all of the
drawbacks mentioned above. Therefore what is needed is an
electrochemical sensor that incorporates an interference-correcting
electrode to minimize the interference caused by oxidizable
substances present in the sample fluid. What is further needed is
an electrochemical sensor whose response is substantially
independent of the hematocrit of the sample fluid. What is still
further needed is an electrochemical sensor which requires less
sample volume than previously required by the prior art. Yet, what
is still further needed is an electrochemical sensor which has a
wide linear measurement range; that is, a sensor having a reduced
or negligible interference effect and useable over a wider glucose
concentration.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved
electrochemical sensor which combines an enzyme and a mediator. It
is a further object of the present invention to provide an
electrochemical sensor that incorporates an interference-correcting
electrode to minimize the interference caused by oxidizable
substances present in the sample fluid. It is a further object of
the present invention to provide an electrochemical sensor whose
response is substantially independent of the hematocrit levels of
the sample fluid. It is still another object of the present
invention to provide an electrochemical sensor which requires less
sample volume than previously required by the prior art. It is yet
another object of the present invention to provide an
electrochemical sensor which has a wide linear measurement
range.
The present invention achieves these and other objectives by
providing an electrochemical sensor which requires a smaller sample
size and compensates for interference from oxidizable species in
the sample and from varying hematocrit levels. The present
invention has a laminated, elongated body having a sample fluid
channel connected between an opening on one end of the laminated
body and a vent hole spaced from the opening. Within the fluid
channel lies at least two working electrodes and a reference
electrode. The arrangement of the two working electrodes and the
reference electrode is not important for purposes of the results
obtained from the electrochemical sensor. The working electrodes
and the reference electrode are each in electrical contact with
separate conductive conduits, respectively. The separate conductive
conduits terminate and are exposed for making an electrical
connection to a reading device on the end opposite the open channel
end of the laminated body.
The laminated body has a base insulating layer made from a plastic
material. Several conductive conduits are delineated on the base
insulating layer. The conductive conduits may be deposited on the
insulating layer by screen printing, by vapor deposition, or by any
method that provides for a conductive layer which adheres to the
base insulating layer. The conductive conduits may be individually
disposed on the insulating layer, or a conductive layer may be
disposed on the insulating layer followed by etching/scribing the
required number of conductive conduits. The etching process may be
accomplished chemically, by mechanically scribing lines in the
conductive layer, by using a laser to scribe the conductive layer
into separate conductive conduits, or by any means that will cause
a break between and among the separate conductive conduits required
by the present invention. The preferred conductive coatings are
gold film or a tin oxide/gold film composition. It should be
pointed out that although the same electrically conducting
substance (gold film or tin oxide/gold film) after scoring is used
as conducting material for both working electrodes and the
reference electrode, this material itself cannot function as a
reference electrode. To make the reference electrode work, there
must be a redox reaction (e.g., Fe(CN).sub.6.sup.3-
+e.sup.-.fwdarw.Fe(CN).sub.6.sup.4-) at the electrically conducting
material when a potential is applied. Therefore, a redox mediator
must be present at the conducting material used for the reference
electrode.
On top of the base insulating layer and the conductive conduits,
the laminated body has a first middle insulating layer containing
cutouts for at least two working electrodes and a reference
electrode. One of the working electrodes and reference electrode
may share the same cutout, provided that the electrode material
(described later) disposed in the cutout is scored to isolate the
working electrode from the reference electrode. Where three cutouts
are used, each cutout corresponds to and exposes a small portion of
a single conductive conduit. The cutouts for the working electrodes
are substantially the same size. The cutout for the reference
electrode may be the same or different size as the cutouts for the
working electrodes. The placement of all of the cutouts are such
that they will all co-exist within the sample fluid channel
described above. This first middle insulating layer is also made of
an insulating dielectric material, preferably plastic, and may be
made by die cutting the material mechanically or with a laser and
then fastening the material to the base layer. An adhesive, such as
a pressure-sensitive adhesive, may be used to secure the first
middle insulating layer to the base layer. Adhesion may also be
accomplished by ultrasonically bonding the first middle layer to
the base layer. The first middle insulating layer may also be made
by screen printing the first middle insulating layer over the base
layer.
The thickness of the first middle layer must be of sufficient
thickness for loading a sufficient amount of electrode material for
use as an electrochemical sensor. Each cutout contains electrode
material. The electrode material has a redox mediator with at least
one of a stabilizer, a binder, a surfactant, and a buffer. At least
one of the cutouts also contains an enzyme capable of catalyzing a
reaction involving a substrate for the enzyme. The redox mediator
is capable of transferring electrons between the enzyme-catalyzed
reaction and the working electrode.
The laminated body also has a second middle insulating layer on top
of the first middle layer. The second middle layer is also made of
a plastic insulating material and creates the sample fluid channel
of the laminated body. It contains a U-shaped cutout on one end
which overlays the cutouts on the first middle layer with the open
end corresponding to the open end of the laminated body described
earlier.
The laminated body of the present invention has a top layer with a
vent opening. The vent opening is located such that at least a
portion of the vent opening overlays the bottom of the U-shaped
cutout of the second middle insulating layer. The vent allows air
within the sample fluid channel to escape as the sample fluid
enters the open end of the laminated body. The sample fluid
generally fills the sample fluid channel by capillary action. In
small volume situations, the extent of capillary action is
dependent on the hydrophobic/hydrophilic nature of the surfaces in
contact with the fluid undergoing capillary action. This is also
known as the wetability of the material. Capillary forces are
enhanced by either using a hydrophilic insulating material to form
the top layer, or by coating at least a portion of one side of a
hydrophobic insulating material with a hydrophilic substance in the
area of the top layer that faces the sample fluid channel between
the open end of the laminated body and the vent opening of the top
layer. It should be understood that an entire side of the top layer
may be coated with the hydrophilic substance and then bonded to the
second middle layer.
The insulating layers of the laminated body may be made from any
dielectric material. The preferred material is a plastic material.
Examples of acceptable compositions for use as the dielectric
material are polyvinyl chloride, polycarbonate, polysulfone, nylon,
polyurethane, cellulose nitrate, cellulose propionate, cellulose
acetate, cellulose acetate butyrate, polyester, acrylic, and
polystyrene.
The number of cutouts in the first middle layer can be one, two and
three or more. To use only one cutout, the single cutout must
expose portions of two conductive conduits. The electrode material
within the single cutout is scored in the middle to separate it
into two parts; one acting as the working electrode and the other
acting as the reference electrode. Such an arrangement allows for
testing a smaller sample volume compared to two or three cutout
embodiment. However, this embodiment lacks the interference and
hematocrit correction features of the other embodiments.
An embodiment having two cutouts is an alternative to the single
cutout version. It has one cutout serving as the working electrode
and the other one serving as a reference electrode. Another
embodiment of the two cutout version combines the features of
making the single cutout with that of the two cutout version. One
of the cutouts containing electrode material is scored into two
parts, one part serving as a first working electrode and the second
part serving as the reference electrode. The second cutout serves
as a second working electrode. Such a design is an alternative
embodiment of the preferred embodiment of the present invention.
This version of the two-cutout embodiment has the interference and
hematocrit correction features but also allows for measuring an
even smaller sample volume than that of the three-cutout
embodiment.
In the three-cutout embodiment, two cutouts contain material for
the working electrodes (W1 and W2) and one for the reference
electrode (R). W2 further contains the enzyme capable of catalyzing
a substrate of the enzyme. The three electrodes are positioned and
sized in such a way that the resistance of the fluid sample could
be precisely measured and the possible carry-over from W2 could be
minimized. The possible electrode arrangements within the sample
fluid channel may be W1-W2-R, W1-R-W2, R-W1-W2, W2-W1-R, W2-R-W1,
or R-W2-W1 with the arrangement listed as the arrangement of
electrodes would appear from the open end of the laminated body to
the vent opening. The preferred position was found to be W1-R-W2;
that is, as the sample fluid entered the open end of the laminated
body, the fluid would cover W1 first, then R, then W2. The
preferred position allows for the precise measurement of blood
sample resistance. This is necessary for good correlation between
the resistance and hematocrit level in the blood sample.
As mentioned earlier, oxidizable interferants such as ascorbic
acid, uric acid and acetaminophen, to name a few, cause inaccurate
readings in the output of an electrochemical biosensor. The present
invention negates this effect by subtracting the current response
at W1 (first working electrode) from the current response from W2
(second working electrode) to calculate the enzyme concentration in
the sample fluid. This is achieved by maintaining the surface area
of W1 substantially equal to the surface area of W2. Also important
is the composition of the reagents disposed on W1 and W2. The
reagents are designed to have a minimal effect on the response of
the interferences which also contributes to the accuracy of the
analyte measurement.
The hematocrit interference is reduced by using a two-step process.
First, the resistance (r-value) between W1 (first working
electrode) and R (reference electrode) is measured. The r-value is
then used to estimate the hematocrit level in the sample fluid. The
following equation represents this relationship:
where
r is resistance value measured in Ohms or Kilo-Ohms
H is hematocrit level
k.sub.1 is a constant equal to 4.6 (r measured in Kilo-Ohms)
Second, the hematocrit level value is then used to mathematically
correct the enzyme concentration reading obtained from above. The
following equation represents the calculation performed using the
calculated hematocrit level from Eq. (1):
C.sub.corr =C.sub.mea /(k.sub.2 +k.sub.3 C.sub.mea +(k.sub.4
+k.sub.5 C.sub.mea)(1-H)) Eq. (2)
where
C.sub.corr is the corrected analyte concentration
C.sub.mea is the measured analyte concentration
k.sub.2 is a constant equal to 1.03
k.sub.3 is a constant equal to -0.003
k.sub.4 is a constant equal to -0.1
k.sub.5 is a constant equal to 0.0054
H is the calculated hematocrit level from Eq. (1)
The constant values above have been determined for the preferred
embodiment of the present invention. Varying the surface area of
the electrode areas and the formulations of the reagents may
require one skilled in the art to calculate new values for
constants k.sub.1 -k.sub.5 in order to more accurately determine
corrected glucose concentration.
All of the advantages of the present invention will be made clearer
upon review of the detailed description, drawings and appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of the present invention showing the
open end, the vent and the electrical contact points of the
laminated body.
FIG. 2 is an exploded, perspective view of the present invention
showing the various layers of the laminated body.
FIGS. 3A, 3B, 3C, and 3D are top views of a strip of each layer of
the present invention showing the patterns for making multiple
sensors of the present invention.
FIG. 3E is a top view of a segment of the laminated strip of the
present invention showing the patterns for making multiple sensors
of the present invention.
FIGS. 4A and 4B are graphs showing the effect of hematocrit on the
concentration response of the present invention in normal and high
concentrations of blood glucose.
FIG. 5 is a correlation of sample volume on the concentration
response of the present invention.
FIG. 6 is a correlation curve of the concentration readings using
sensors of the present invention versus the concentration readings
of obtained on the same samples using a YSI glucose analyzer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The preferred embodiment of the present invention is illustrated in
FIGS. 1-6. FIG. 1 shows a sensor 10 of the present invention.
Sensor 10 has a laminated body 100, a fluid sampling end 110, an
electrical contact end 120, and a vent opening 52. Fluid sampling
end 110 includes a sample fluid channel 112 between a sampling end
aperture 114 and vent opening 52. Electrical contact end 120 has at
least three discreet conductive contacts 122, 124 and 126.
Referring now to FIG. 2, laminated body 100 is composed of a base
insulating layer 20, a first middle layer 30, a second middle layer
40, and a top layer 50. All layers are made of a dielectric
material, preferably plastic. Examples of a preferred dielectric
material are polyvinyl chloride, polycarbonate, polysulfone, nylon,
polyurethane, cellulose nitrate, cellulose propionate, cellulose
acetate, cellulose acetate butyrate, polyester, acrylic and
polystyrene. Base insulating layer 20 has a conductive layer 21 on
which is delineated a first conductive conduit 22, a second
conductive conduit 24 and a third conductive conduit 26. Conductive
conduits 22, 24 and 26 may be formed by scribing or scoring the
conductive layer 21 as illustrated in FIG. 2 or by silk-screening
the conductive conduits 22, 24 and 26 onto base layer 20. Scribing
or scoring of conductive layer 21 may be done by mechanically
scribing the conductive layer 21 sufficiently to create the three
independent conductive conduits 22, 24 and 26. The preferred
scribing or scoring method of the present invention is done by
using a carbon dioxide (CO.sub.2) laser, a YAG laser or an eximer
laser. An additional scoring line 28 (enlarged and not to scale;
for illustrative purposes only) may be made, but is not necessary
to the functionality of sensor 10, along the outer edge of base
layer 20 in order to avoid potential static problems which could
give rise to a noisy signal. Conductive layer 21 may be made of any
electrically conductive material, preferably gold or tin
oxide/gold. A useable material for base layer 20 is a tin
oxide/gold polyester film (Cat. No. FM-1) or a gold polyester film
(Cat. No. FM-2) sold by Courtaulds Performance Films, Canoga Park,
Calif.
First middle layer 30 has a first electrode cutout 32 which exposes
a portion of first conductive conduit 22, a second electrode cutout
34 which exposes a portion of second conductive conduit 24 and a
third electrode cutout 36 which exposes a portion of third
conductive conduit 26. First layer 30 is made of a plastic
material, preferably a medical grade one-sided tape available from
Adhesive Research, Inc., of Glen Rock, Pa. Acceptable thickness of
the tape for use in the present invention are in the range of about
0.003 in. (0.76 mm) to about 0.005 in. (0.127 mm). One such tape,
Arcare.RTM.7815, was preferred because of its ease of handling and
it showed good performance in terms of its ability to hold a
sufficient quantity of chemical reagents and to promote a favorable
blood flood speed (capillary action) through sample fluid channel
112 of sensor 10. It should be understood that the use of a tape is
not required. A plastic insulating layer may be coated with a
pressure sensitive adhesive, or may be ultrasonically-bonded to
base layer 20, or may be silk-screened onto base layer 20 to
achieve the same results as using the polyester tape mentioned.
The three cutouts 32, 34 and 36 define electrode areas W1, R and
W2, respectively, and hold chemical reagents forming two working
electrodes and one reference electrode. Typically, electrode area R
must be loaded with a redox reagent or mediator to make the
reference electrode function. If R is not loaded with a redox
reagent or mediator, working electrodes W1 and W2 will not work.
Electrode areas W1 and R are loaded preferably with the same
chemical reagent to facilitate the resistance measurement described
earlier. The reagents preferably contain an oxidized form of a
redox mediator, a stabilizer, a binder, a surfactant, and a buffer.
Typically, the redox mediator may be at least one of ferrocene,
potassium ferricyanide and other ferrocene derivatives. The
preferred stabilizer is polyethylene glycol, the preferred binder
is methyl cellulose, the preferred surfactant is
t-octylphenoxypolyethoxyethanol, and the preferred buffer is a
citrate buffer. Electrode area W2 is preferably loaded with the
same chemical reagents loaded into electrode areas W1 and R but
with the addition of an enzyme capable of catalyzing a reaction
involving a substrate for the enzyme or a substrate catalytically
reactive with an enzyme and a mediator capable of transferring
electrons transferred between the enzyme-catalyzed reaction and the
working electrode to create a current representative of the
activity of the enzyme or substrate and representative of the
compound.
The cutouts and electrode areas of first layer 30 are positioned
relative to each other and to the flow of the sample fluid in
sample fluid channel 112 such that the resistance of the sample
fluid may be precisely measured and the possible carryover from
electrode area W2 to electrode area W1 could be minimized. Using
fluid sample end 110 of sensor 10 as a reference point, the
arrangements of the electrode areas could be W1-W2-R, W1-R-W2,
R-W1-W2, W2-W1-R, W2-R-W1, or R-W2-W1. The preferred position was
found to be W1-R-W2.
Second middle layer 40 has a U-shaped channel cutout 42 located at
second layer sensor end 41. The length of channel cutout 42 is such
that when second middle layer 40 is layered on top of first middle
layer 30, electrode areas W1, W2 and R are within the space defined
by channel cutout 42. The thickness of second middle layer 40 was
found to be critical for the speed of the sample fluid flow into
sample fluid channel 112, which is filled by capillary action of
the sample fluid.
Top layer 50, which is placed over second middle layer 40, has a
vent opening 52 spaced from fluid sample end 110 of sensor 10 to
insure that sample fluid in fluid channel 112 will completely cover
electrode areas W1, W2 and R. Vent opening 52 is placed in top
layer 50 so that it will align somewhat with the bottom of channel
cutout 42 of second middle layer 40. Preferably, vent opening 52
will expose a portion of and partially overlay the bottom of the
U-shaped cutout 42 of second middle layer 40.
Preparation of Reagents 1 & 2
Reagents 1 and 2 comprise the oxidized form of a redox mediator, a
stabilizer, a binder, a surfactant, and a buffer. Reagent 2, in
addition, contains an enzyme. The oxidized form of the redox
mediator, potassium ferricyanide, was found to be stable in the
matrices. The quantity used in the formulation must be sufficient
to attain a workable linear range. The enzyme must also have
sufficient activity, purity and stability. A commercially available
glucose oxidase may be obtained from Biozyme, San Diego, Calif. as
Cat. No. G03A, about 270 U/mg. The stabilizer must be sufficiently
water-soluble and be capable of stabilizing both the mediator and
the enzyme. The binder should also be capable of binding all other
chemicals in the reagents in electrode areas W1, W2 and R to the
conductive surface/layer 21 of base layer 20. The preferred
stabilizer is polyethylene glycol (Cat. No. P4338, Sigma Chemicals,
St. Louis, Mo.). The preferred binder is Methocel 60 HG (Cat. No.
64655, Fluka Chemical, Milwaukee, Wis.). The buffer solution must
have sufficient buffer capacity and pH value to optimize the enzyme
reaction. A0.05 M citrate buffer is preferred. The surfactant is
necessary to facilitate dispensing of Reagents 1 and 2 into cutouts
32, 34 and 36 of middle layer 30 as well as for quickly dissolving
the dry chemical reagents. The amount and type of surfactant is
selected to assure the previously mentioned functions and to avoid
a denaturing effect on the enzyme. The preferred surfactant is
Triton X-100. The reagents are prepared as follows:
Reagent 1
Step 1: Prepare 50mM citrate buffer (pH 5.7) by dissolving 0.1512
grams citric acid and 1.2580 grams sodium citrate in 100 ml of
deionized water.
Step 2: Prepare a 1% methocel 60HG solution by stirring 1 gram of
methocel in 100 ml of citrate buffer from Step 1 for 12 hours.
Step 3: Add 0.3 ml of 10% Triton X-100 into the methocel
solution.
Step 4: Add 2.5 grams of polyethylene glycol into the solution from
Step 3.
Step 5: While stirring, add 1 gram of potassium ferricyanide to the
solution from Step 4.
Reagent 2
Step 1-Step 4: same steps as Reagent 1.
Step 5: While stirring, add 6.5 grams potassium ferricyanide to the
solution of Step 4.
Step 6: Add 1.0 gram of glucose oxidase to the solution of Step 5
and stir for 10 minutes or until all solid materials are completely
dissolved.
Electrode Construction
A piece of a gold or tin oxide/gold polyester film available from
Courtaulds Performance Films is cut to shape, as illustrated in
FIG. 2, forming base layer 20 of sensor 10. A CO.sub.2 laser was
used to score the gold or tin oxide/gold polyester film. As
illustrated in FIG. 2, the film was scored by the laser such that
three electrodes at sample fluid end 110 and three contact points
122, 124 and 126 were formed at electrical contact end 120. The
scoring line is very thin but sufficient to create three separate
electrical conductors. A scoring line 28 can be made, but is not
necessary, along the outer edge of base layer 20 to avoid potential
static problems which could cause a noisy signal from the finished
sensor 10.
A piece of one-sided adhesive tape is then cut to size and shape
forming first middle layer 30 so that it will cover a majority of
the conductive layer 21 of base layer 20 except for exposing a
small electrical contact area illustrated in FIG. 1. Three
rectangular, square or circular cutouts 32, 34 and 36 of
substantially equal size are punched by CO.sub.2 laser (25W laser
available from Synrad, Inc., San Diego, Calif.). Cutouts 32, 34 and
36 define the electrode areas W1, W2 and R which hold chemical
reagents. The size of the cutouts is preferred to be made as small
as possible in order to make the fluid sample channel 112 of sensor
10 as short as possible while still being capable of holding
sufficient chemical reagent to function properly. The preferred
hole size for the present invention has a typical dimension of
about 0.033 in. (0.84 mm) by about 0.043 in. (1.09 mm). As
illustrated in FIG. 2, cutouts 32, 34 and 36 are aligned with each
other and having a spacing of about 0.028 in. (0.71 mm) between
them. The rectangular cutouts are for illustrative purposes only.
It should be understood that the shape of the cutouts is not
critical provided that the size of the cutouts is big enough to
hold sufficient chemical reagents for the electrodes to function
properly but small enough to allow for a reasonably small sample
channel. As noted earlier, changing the shape of the cutouts or the
surface area of the cutouts may require changing the constant
values k.sub.1 -k.sub.5 for Eq. 1 and Eq. 2. As stated previously,
the preferred arrangement of the electrodes formed in cutouts 32,
34 and 36 is W1 (working electrode 1), R (reference electrode) and
W2 (working electrode 2).
0.4 microliters of Reagent 1 is dispensed into electrode areas W1
and R. Reagent 1 is a mixture of a redox mediator, a stabilizer, a
binder, a surfactant, and a buffer. The preferred mixture for
Reagent 1 is made by mixing the following components in the
described percentages (W/W %): about 1% potassium ferricyanide,
about 2.5% polyethylene glycol, about 1% methocel 60 HG, about
0.03% Triton X-100 and about 0.05 M citrate buffer (pH 5.7). 0.4
microliters of Reagent 2 is dispensed into electrode area W2.
Reagent 2 is a mixture similar to that of Reagent 1 but with the
addition of an enzyme capable of catalyzing a reaction involving a
substrate of the enzyme. The preferred enzyme is glucose oxidase.
The preferred mixture for Reagent 2 is made by mixing the following
percentages (W/W %) of the following ingredients: about 6.5%
potassium ferricyanide, about 2.5% polyethylene glycol, about 1%
methocel 60 HG, about 0.03% Triton X-100, about 0.05 M citrate
buffer (pH 5.7), and about 1% glucose oxidase. After the addition
of the reagents, the device was dried for about 2 minutes at
55.degree. C. in an oven. After drying, a piece of double-sided
tape available from Adhesive Research was fashioned into second
middle layer 40 with U-shaped channel 42. Second middle layer 40 is
then layered onto first middle layer 30. As mentioned earlier, this
second middle layer 40 serves as a spacer and defines the size of
the fluid sample channel 112. Its width and length is optimized to
provide for a relatively quick moving fluid sample. The preferred
size of U-shaped channel 42 is about 0.063 in. (1.60 mm) wide by
about 0.248 in. (6.30 mm) long.
A piece of a transparency film (Cat. No. PP2200 or PP2500 available
from 3 M) is fashioned into top layer 50. A rectangular vent hole
52 is made using the CO.sub.2 laser previously mentioned. The
preferred size of vent hole 42 is about 0.075 in. (1.91 mm) by
about 0.059 in. (1.50 mm). Vent hole 52 is located approximately
0.130 in. (3.3 mm) from fluid end 110 of sensor 10. Top layer 50 is
aligned and layered onto second middle layer 40 to complete the
assembly, as illustrated in FIG. 1, of sensor 10.
Although the description of electrode construction above describes
construction for a single sensor, the design and materials used are
ideal for making multiple sensors from one piece of each layer
material as shown in FIGS. 3A-3E. This would be accomplished by
starting with a relative large piece of base layer 20 having
conducting layer 21 thereon. A plurality of scored lines are made
into conductive layer 21 such that a repetitive pattern, as
illustrated in FIG. 3A, is created using the preferred scribing
method described previously whereby each pattern will eventually
define the three conductive paths 22, 24 and 26 for each sensor.
Similarly, a large piece of first middle layer 30, which is
illustrated in FIG. 3B and which also has a plurality of cutouts
32, 34, and 36 in a repetitive pattern, is sized to fit over base
layer 20 in such a way that a plurality of sensors 10 will be had
when completed. The size of each cutout and the electrode material
disposed in the plurality of electrode areas W1, R and W2 are
similar to that disclosed above. After disposing Reagents 1 & 2
in their respective cutouts and dried, a large piece of second
middle layer 40 having a plurality of elongated cutouts 42 and
illustrated in FIG. 3C is layered onto first middle layer 30 such
that each elongated cutout 42 of second middle layer 40 contains
corresponding cutouts 32, 34 and 36 of first middle layer 30. A
comparably-sized top layer 50 having a plurality of vent openings
52 in a repetitive pattern, as shown in FIG. 3D, is layered onto
second middle layer 40. FIG. 3E is a top view of the combined
layers. The laminated strip created by the four layers 20, 30, 40
and 50 has a plurality of sensors 10 that can be cut from the
laminated strip. The laminated strip is cut longitudinally along
line A-A' at fluid sampling end 210 to form a plurality of sampling
apertures 114 and longitudinally along line B-B' at electrical
contact end 220 to form a plurality of conductive contacts 122, 124
and 126. The laminated strip is also cut at predetermined intervals
along line C-C' forming a plurality of individual sensors 10.
Shaping of the fluid sampling end 120 of each sensor 10, as
illustrated in FIG. 1, may be performed if desired. It should be
understood by those skilled in the art that the order in which the
laminated strip can be cut is not important. For instance, the
laminated strip may be cut at the predetermined intervals (C-C')
and then the cuts along A-A' and B-B' can be made to complete the
process.
The following examples illustrate the unique features of the
present invention which includes the compensation for varying
hemotacrit levels by measuring sample fluid resistance and
nullification of the interference effects of oxidizable species
present in the sample fluid. All sensors of the present invention
were tested on a breadboard glucose meter manufactured by Nova
Biomedical Corporation of Waltham, Mass. A potential of 0.35 Volts
was applied across the working electrodes and the reference
electrode and the resultant current signals were converted to
glucose concentrations in accordance with the disclosure of the
present invention. The readings were compared to readings (control
readings) obtained on the same samples using YSI Glucose Analyzer
(Model 2300) available from Yellow Springs Instruments, Inc.,
Yellow Springs, Ohio.
EXAMPLE 1
Demonstration of Hematocrit Compensation
The unique design of the present invention makes it possible to
measure the resistance of the fluid sample. This is achieved by
applying the same reagent, Reagent 1, to the reference electrode R
and the first working electrode W1. The chemical reagents used in
Reagent 1 are critical for accurate measurement of the resistance.
Reagent 1 can not contain a large amount of salts or any glucose
oxidase. Otherwise, the resulting resistance would not be accurate
and would be glucose dependent. For proper functioning of the
present invention, it should be noted that a minimum amount of a
mediator such as potassium ferricyanide for the reference electrode
is essential.
Resistance of a sample fluid, in this case blood samples, between
W1 and R is measured at any time, preferably 20 seconds after a
reading device (Nova glucose meter) is triggered by the blood
samples. Blood samples with different hematocrit levels were
prepared by spinning a whole blood sample and recombining plasma
and red blood cells in varying ratios. Hematocrit levels were
measured with a micro hematocrit centrifuge. Concentrations of
glucose in the various samples were measured by sensors of the
present invention (C.sub.mea) and by a YSI blood glucose analyzer
(the control), Model 2300, Yellow Springs Instruments, Inc., Yellow
Springs, Ohio. Equations (1) and (2), previously mentioned, were
used to calculate the corrected glucose concentration (C.sub.corr)
measured by sensors of the present invention to demonstrate the
hemotacrit compensation feature of the present invention. The data
obtained was plotted and FIGS. 4A and 4B show two graphs
representing the percent correlation of the readings obtained using
sensors of the present invention with the Nova glucose meter to the
readings obtained for the samples using the YSI blood glucose
analyzer at low and high levels of glucose in samples with varying
hematocrit levels.
EXAMPLE 2
Demonstration of Interference Free Feature
The unique design of the present invention makes it possible to
eliminate interference from oxidizable substances such as ascorbic
acid, acetaminophen, uric acid, and other possible interferants
present in the sample. This is achieved by subtracting the response
obtained from W1 from the response obtained at W2, and is
represented by the following equation:
where
Iw.sub.2 is the current at W2 (second working electrode)
Iw.sub.1 is the current at W1 (first working electrode)
I is the difference between W2 and W1 and represents the current
due to oxidation of the mediator of its reduced form, which is
proportional to the glucose concentration in the sample
Because W1 and W2 have the same surface area, the potential
interference present in the sample fluid should give relatively
identical signals from each working electrode. Even though W1 and
W2 had different reagents, it was found that there was no
remarkable difference in the response to the interference. Thus,
the difference in current response obtained in blood samples was
due to the glucose present in the samples. This was tested by
spiking normal and high glucose blood samples with 1 mM and 5 mM
ascorbic acid, acetaminophen and uric acid. Table 1 shows the
percentage response change of the readings obtained with sensors of
the present invention and various commercially available sensors
(referred to as Strip 1, Strip 2, Strip 3, and Strip 4) in blood
samples having a concentration of 100 mg/dL glucose and 300 mg/dL
upon addition of the interferents.
TABLE 1 Response change (%) Upon Addition of Interferent mM Nova
Strip 1 Strip 2 Strip 3 Strip 4 100 mg/dL glucose: ascorbic acid 0
0 0 0 0 0 1 4 26.7 19.8 21.3 6 5 4.5 133.6 115.8 151.5 Error 300
mg/dL glucose:ascorbic acid 0 0 0 0 0 0 1 -0.9 7.2 14 14.7 -4.3 5
-0.5 Hi 107.3 Hi Error 100 mg/dL glucose:acetaminophen 0 0 0 0 0 0
1 3.0 34.7 5* 7 50 -8.2 5 3.4 90.8 38 136 -13.4 300 mg/dL
glucose:acetaminophen 0 0 0 0 0 0 1 -3.4 20 3.5 18 -7.2 5 -3.5 39
8.0 23.7 -17.5 100 mg/dL glucose:uric acid 0 0 0 0 0 0 1 -2.7 32 35
35 -1.3 300 mg/dL glucose:uric acid 0 0 0 0 0 0 1 -4.5 10 15 7
-1
From the test data, one observes that the readings obtained from
sensors of the present invention show essentially no change in the
presence of 1 mM and 5 mM ascorbic acid and acetaminophen, and 1 mM
uric acid. All commercially available sensors except one, Strip 4,
suffer from serious interference. Strip 4 showed an "error" for 5
mM ascorbic acid. At concentrations of 300 mg/dL glucose, sensors
of the present invention also showed no interference (response
change of less than 5%) upon spiking the samples with 1 mM and 5 mM
ascorbic acid. The commercially available sensors showed about 7%
to about 15% response increase for 1mM ascorbic acid spiked
samples, and showed a "Hi" reading for 5 mM ascorbic acid spiked
samples. Strip 4 again showed an "error" for 5 mM ascorbic acid
spiked samples. In samples containing acetaminophen and uric acid,
all commercially available strips showed varying degrees of error
except for Strip 4 in samples containing uric acid.
EXAMPLE 3
Demonstration of Minimum Sample Volumes Feature
The unique design of the present invention enables the measurement
of sample sizes smaller than which have heretofore been possible.
Blood samples are applied to the sensors and the samples travel
along the fluid sample channel to the venting hole. The blood
volume required for measurement of blood glucose is determined by
the channel volume. The calculated volume for the present invention
is 1.44 microliters. In order to test the volume effect on sensor
response, different blood sample volumes were applied to the
sensors and the resulting concentration readings were plotted
against volume. The test data is shown in FIG. 5.
Sensors of the present invention show no dependence of the response
on the sample volume if the volume is above 1.5 microliters. It was
found that sensors of the present invention still gave reasonable
readings on sample sizes as low as 1.0 microliters. This is
possible because the hydrophilic character of Reagent 1 applied to
W1 and R, and Reagent 2 applied to W2 permitted the sample to cover
the electrode areas even though the blood volume did not fill the
entire sample channel.
EXAMPLE 4
Demonstration of Wide Linear Range and Precision Feature
A sample of venous blood was collected and separated into several
aliquots. Each aliquot was spiked with different glucose
concentrations ranging from 35 to 1000 mg/dL. The aliquots were
each measured with a YSI glucose analyzer and then with sensors of
the present invention using the Nova glucose meter. Sensors of the
present invention show a linear relationship of current response
vs. glucose concentration from 35 to 1000 mg/dL. The concentration
readings were plotted against the concentration values obtained
using the YSI meter (the control) and are illustrated in FIG.
6.
A regression coefficient of 0.9988 indicated a near perfect match
with the readings obtained with the YSI blood glucose analyzer. The
same aliquots were tested using four different
commercially-available sensors with their accompanying meters. The
commercially-available sensors showed a linear response only up to
about 600 mg/dL. Above the 500-600 mg/dL range, all commercially
available sensors displayed "Hi" as the test result.
The precision of the sensors of the present invention was
investigated at the same glucose level range from about 35 to 1000
mg/dL. Four different batches of sensors of the present invention
were used in the precision tests. Typically, the relative standard
deviation was about 9.5%, 5.0%, 3.5%, 2.9%, and 2.6% for samples
containing 35, 100, 200, 500, and 1000 mg/dL levels of glucose,
respectively.
* * * * *